Novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers

ABSTRACT

Novel Hydrazone-based Fluorescent and Pro-Fluorescent Reagents and Linkers, including conjugationally extended hydrazine compositions, fluorescent hydrazone compositions, methods of the formation of hydrazones from the reaction of conjugationally extended hydrazines with conjugationally extended carbonyls, and methods of their use in assays systems are described. Use of these conjugationally compositions for direct colorimetric and fluorometric assays wherein a chromophore or the fluorophore is incorporated into the linker that is positioned between a reactive linking moiety and a biotin molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a regular application claiming priority fromprovisional patent application Ser. No. 60/792,821 and 60/792,822 bothfiled 18 Apr. 2006.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT DISC

None

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to compounds used to label biomoleculesfor diagnostic and therapeutic purposes. In particular, it relates tofluorescent, chromophoric, pro-fluorescent and pro-chromophoriccompounds that may be conjugated to biomolecules such as proteins andnucleic acids. Such compounds may be incorporated into linkers that maybe used to link a ligand to a biomolecular probe allowing quantitationof the ligand bound to that molecular probe.

(2) Description of Related Art

Methods to detect interactions between biomolecules continues to be anarea of active research as new and more sensitive methods are requiredto increase sensitivity, reduce costs and enable new detection methods.One of the most widely used methods is to directly label a biomoleculewith a fluorescent molecule that fluoresces at a desired frequency. Forexample, a fluorescent molecule is modified with a thiol- oramino-reactive moiety such as succinimidyl esters or maleimides thatform a covalent bound in the presence of a sulfhydryl or amine group ofa desired protein. The modified fluorescent molecule is isolated andreacted with the desired protein. The fluorescently labeled protein isthen used to detect a desired target by monitoring the uniquefluorescent frequency of the fluorophore. A variety of fluorophores havebeen modified with these moieties including fluorescein, rhodamine,Texas Red and cyanine dyes, Cy3 and Cy5. Unfortunately, the conjugationmethods often cause quenching and photobleaching of the fluorophore andthere can be interference with the observed signal if the unboundlabeled biomolecule is not removed from the reaction mixture.

Other biomolecules such as nucleic acids such as DNA, RNA,polynucleotide and oligonucleotides have been labeled with fluorophoresis commonly accomplished by incorporating a fluorophore on the basemoiety of a nucleoside triphosphate. These fluorescently labeledtriphosphates are added to the polymerase chain reaction (PCR) orreverse transcription reaction wherein the labeled nucleoside isincorporated in the amplicon yielding a fluorescently labeledpolynucleotide. These fluorescently labeled polynucleotides are probedusing oligonucleotide microarrays identifying sequences present in thetarget. Unfortunately, the fluorophores used for labeling thesebiomolecules are not often stable to these synthesis conditions. Inaddition, the long-term stability of conjugates are low due tophotobleaching, consequently, retention of the fluorescent signal isdifficult when archiving microarrays.

A variety of references cite the use of fluorescent hydrazides,thiosemicarbazides and hydrazides to react with aldehydes on biologicalmolecules for the detection of the aldehydes. For example Ahn et al. (B.Ahn, S. G. Rhee and E. R. Stadtman, Anal. Biochem. 161:245 (1987)describe the use of fluorescein hydrazide and fluoresceinthiosemicarbazide for the fluorometric determination of protein carbonylgroups and for the detection of oxidized proteins on polyacrylamidegels. Proudnikov and Mirzabekov (Nucl. Acids Res. 24:4535 (1996))describe labeling of DNA and RNA to identify acid-induced depurinationthat results in production of aldehyde moieties detected by reaction offluorescent labels containing hydrazide groups in the presence of sodiumcyanoborohydride. Others have labeled the reducing end ofpolysaccharides with fluorescent hydrazides. These methods are used todetect aliphatic aldehyde groups on biomolecules. In each of thereferences the fluorescent moiety is incorporated on the hydrazine orhydrazide that forms a hydrazone on reaction with the aldehyde presenton the biomolecule.

It has been documented that hydrazones formed between certain aromaticaldehydes and aromatic hydrazines and not aromatic hydrazides oraromatic thiosemicarbazides form fluorescent molecules (J. Wong and F.Bruscato, Tet. Lett. 4593, 1968). It has also been reported thathydrazones formed specifically from 2-substituted aldehyde heterocyclesand 2-substituted hydrazine heterocycles become fluorescent on chelationto zinc (D. E. Ryan, F. Snape and M. Winpe, Anal. Chim. Acta 58:101,1972).

Schwartz et al. (U.S. Pat. No. 5,420,285; U.S. Pat. No. 5,753,520; U.S.Pat. No. 5,420,285; J. Nucl. Med. 31(12):2022, 1990 and Bioconjug. Chem.2(5):333, 1991) describe the preparation of succinimidyl6-hydraziniumnicotinate hydrochloride for the one-step modification ofamino groups on proteins and other molecules to incorporatepyridylhydrazine moieties on proteins for the specific purpose ofbinding technetium-99m for in vivo diagnostic purpose. SubsequentlySchwartz (U.S. patent application, titled: Functional OligonucleotideModification Reagents and Uses Thereof, filed Aug. 1, 2000) describenovel oligonucleotide aldehyde and hydrazine phosphoramidite reagentsfor incorporation of aldehydes and hydrazines on syntheticoligonucleotides including aromatic and heteroaromatic aldehydes andhydrazines. Triphosphates incorporating both aromatic hydrazine andaromatic aldehydes have been described by Schwartz and Hogrefe (U.S.Pat. No. 6,686,461).

Cytidine and deoxycytidine moieties in polynucleotides can betransformed into 4-N-aminocytidine (4-hyd-C), an aromatic hydrazine, bytreatment with hydrazine/bisulfite at neutral pH. Nitta et al. Eur. J.Biochem. 157(2):427, 1986 has described crosslinking between 16Sribosomal RNA and protein S4 in E. coli ribosomal 30S subunits effectedby treatment with bisulfite/hydrazine and bromopyruvate. Also Musso etal., (U.S. Pat. No. 5,130,466) describe labeling of 4-N-aminocytidinemoieties on hydrazine/bisulfite treated DNA to yield a fluorescentlylabeled polynucleotide. Bittner et al. (U.S. Pat. No. 5,491,224) alsodescribe the labeling of transaminated DNA with fluorescent moietiespossessing moieties that react with the transaminated cytosine such asfluorophores possessing succinimidyl esters.

In all of the aforementioned references the biomolecule is fluorescentlylabeled with a fluorescent molecule. Unfortunately as previously statedthe processes or methods used to prepare the conjugate can often timescause quenching or photobleaching of the fluorophore. In addition,during use the unbound fluorescently labeled conjugate must be removedto obtain an accurate fluorescent signal.

Therefore, there is a need in the field for a fluorescent label that isresistant to reaction conditions necessary for producing a labeledbiomolecule and does not require removal of the unbound fluorescentlylabeled biomolecule from the detection reaction mixture to obtain aaccurate and/or quantitative signal. There is also a need forfluorophores that may be formed under standard assay conditions frompro-fluorophores which, are stable under various laboratory conditionsand by a reaction that is highly specific and efficient.

To date the most commonly used method to link, immobilize and detectbiomolecules is the biotin/streptavidin ligand/receptor couple. Biotin(FIG. 1) is a small molecule, MW 250, that binds to streptavidin with anassociation constant of 10¹⁵. The extremely high binding constant andfast kinetics of binding and the stability of avidin under a variety ofconditions make this an ideal ligand/receptor pair for these purposes.Biotin has been modified to include amino, thiol and carbohydratereactive moieties, i.e. succinimidyl ester, maleimido and hydraziderespectively, to allow easy incorporation into a large variety ofbiomolecules. To accomplish detection of an analyte, biotin isconjugated to a probing biomolecule such as an antibody or anoligonucleotide. Following binding of the biotinylated biomolecule toits receptor or complement, an avidin/reporter conjugate such as anavidin/fluorophore conjugate or a avidin/reporter enzyme conjugate isadded and allowed to bind to biotinylated probe and visualized byfluorescence detection or addition of a substrate that emits light orprecipitates a colored insoluble product on enzymatic processing(Heitzmann H., Richards F. M., Proc. Natl. Acad. Sci. USA 71:3537-3541,1974; Diamandis E. P., Christopoulos T. K., Clin. Chem. 37:625-636,1991; Wilchek M. Methods Enzymol Vol. 184, 1990; Savage, M. D. et al.,1992 Avidin-Biotin Chemistry: A Handbook. Rockford, Ill.: PierceChemical Co.).

Following conjugation it is important to determine that the probemolecule has been biotinylated and to quantify the number of biotins nowconjugated to the probe molecule. To this end two multi-step indirectassays have been developed. The first assay is the HABA([2-(4′-hydroxyazobenzene)]benzoic acid) assay developed by Green(Green, N. M. Biochem. J., 94, 23c-24, 1965). To quantify biotin labelincorporation, a solution containing the biotinylated protein is addedto a mixture of HABA and avidin. Because of its higher affinity foravidin, biotin displaces the HABA from its interaction with avidin andthe absorption at 500 nm decreases proportionately. By this method, anunknown amount of biotin present in a solution can be evaluated in asingle cuvette by measuring the absorbance of the HABA-avidin solutionbefore and after addition of the biotin-containing sample. The change inabsorbance relates to the amount of biotin in the sample.

The second more sensitive fluorescence-based multi-step assay developedby Molecular Probes (recently acquired by Invitrogen Corporation inCarlsbad, Calif.) is the ‘Fluoreporter Biotin Quantitation Assay’ thatis based on fluorescence resonance energy transfer (FRET) quenchingwherein an avidin molecule is labeled with a fluorophore and its bindingsites are occupied with a fluorescent molecule that quenches thecovalently linked fluorophore until the quencher in the binding site isdisplaced by a higher binding biotin molecule resulting in fluorescenceof the covalently attached fluorophore. While this assay is sensitive to50-100 pmol range it requires many processing steps and a fluorimeter ormulti-well fluorimeter. It is also recommended to digest thebiotinylated protein prior to the assay to expose any stericallyencumbered biotins.

Consequently there is a need in the field for a assay wherein the numberof biotins covalently linked to a biomolecule could be determined bydirect methods such as spectroscopic means.

BRIEF SUMMARY OF THE INVENTION

The present invention provides profluorescent/prochromophoric hydrazineand aldehyde reagent compounds for preparing novel hydrazone-basedfluorescent molecules. More specifically conjugationally extendedprofluorescent/prochromophoric hydrazine compounds of the formula(RR₂)N(H)_(n)(NH₂)_(n), wherein R is independently a substituted orunsubstituted conjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ is independently ahydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, abranched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphaticmoiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; n is 0 when m is andn is 1 when m is 1 may be combined with conjugationally extendedprofluorescent/prochromophoric carbonyl compounds of the formulaO═C(R₁R₂) wherein: R₁ is independently a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ is independently ahydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, abranched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphaticmoiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; n is 0 when m is 2and n is 1 when m is 1 to form fluorescent hydrazone compounds of theformula (RR₂)NN═C(R₁R₂).

In one embodiment the hydrazone compound has the formula:

wherein R₁ (which is R₂) is independently a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ (which is R₃) isindependently a hydrogen, a straight chain aliphatic moiety of 1-10carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclicaliphatic moiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₃ (which is R₄) is Hor OH; R₄ (which is R₆) is H or a nucleic acid moiety; and R₅ (which isR₇) is PO₃ or a nucleic acid moiety.

In another embodiment these novel profluorophore hydrazine and carbonylcompounds may further comprise a linkable moiety at one of the R or R₂positions wherein the linkable moiety is selected from the groupconsisting of an amino reactive moiety, a thiol reactive moiety, anester moiety and a modified carbohydrate monomer moiety.

In yet another embodiment a biomolecule such as for example a nucleicacid, a nucleotide, a protein, an amino acid, a carbohydrate monomer ora polysaccharide is linked to the profluorescent/prochromophorichydrazine and/or profluorescent/prochromophoric carbonyl by a linkablemoiety. If the biomolecule is a nucleic acid it may be DNA, cDNA, RNA,or PNA and can comprise natural or unnatural bases or internucleotidelinkages selected from the group consisting of phosphodiesters,phosphorothioates, phosphoramidites and peptide nucleic acids.

In still another embodiment one or more of theprofluorescent/prochromophoric hydrazine or carbonyl compounds may bebound to a polymer such as poly-lysine, poly-ornithine orpolyethyleneglycol by one or more linkable moieties.

In another aspect of the present invention methods of forming ahydrazone compound are provided by combining the conjugationallyextended profluorescent/prochromophoric hydrazine of formula(RR₂)N(H)_(n)(NH₂) with conjugationally extendedprofluorescent/prochromophoric carbonyl of the formula O═C(R₁R₂) for atime and under conditions that allow hydrazone formation.

In one embodiment of this aspect of the invention the conjugationallyextended profluorescent/prochromophoric hydrazine and/or theconjugationally extended profluorescent/prochromophoric carbonyl mayfurther comprise a linkable moiety at either the R₁ or R₂ position.

In yet another aspect of the invention a method for labeling abiomolecule with a fluorescent hydrazone compound is provided.

In still another aspect the present invention provides oxyamine andaldehyde reagent compounds for preparing novel oxime-based fluorescentmolecules. More specifically conjugationally extendedprofluorescent/prochromophoric oxyamine compound of formula: (R₁R₂)ONH₂are provided wherein: R₁ is a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; and R₂ is a hydrogen,a straight chain aliphatic moiety of 1-10 carbon atoms, a branchedaliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10carbon atoms, a substituted or unsubstituted conjugationally extendedmoiety wherein the unsubstituted conjugationally extended moiety is analkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic orpolyheteroaromatic moiety and wherein the substituted conjugationallyextended moiety may be substituted with any combination of one or moreof the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo,unsubstituted amine and substituted primary, secondary, tertiary andquaternary amine.

In one embodiment a profluorescent/prochromophoric oxyamine compound isprovided wherein R₁ or R₂ further comprise a linkable moiety selectedfrom the group consisting of an amino reactive moiety, a thiol reactivemoiety, an ester moiety and a modified carbohydrate monomer moiety.

In another embodiment a profluorescent/prochromophoric oxyamine compoundis provided wherein the linker further comprises a biomolecule selectedfrom the group consisting of a nucleic acid, a nucleotide, a protein anamino acid, a carbohydrate monomer and a polysaccharide. The nucleicacid may be selected from the group consisting of DNA, cDNA, RNA and PNAand may comprise natural or unnatural bases or internucleotide linkagesselected from the group consisting of phosphodiesters,phosphorothioates, phosphoramidites and peptide nucleic acids.

In another aspect of the invention a spectrophotometrically quantifiablelinker is provided comprising of formula: A-B-C-D wherein A is an amino,thiol or carbohydrate reactive moiety; B is a chromophoric orfluorescent moiety; C is a flexible linker; and D is biotin or areceptor ligand. When A is an amino reactive moiety it may be selectedfrom the group consisting of N-hydroxysuccinimidyl, p-nitrophenyl,pentafluorophenyl and N-hydroxybenzotriazolyl. When A is a thiolreactive moiety it may be selected from the group consisting ofmaleimido, α-haloacetamido and pyridylsulfides. When A is a carbohydratereactive moiety it may be aminooxy. B may be a compound that fluoresces,emits light or precipitates a colored insoluble product on enzymaticprocessing. C is a flexible linker and may be a PEG flexible linkerhaving no less than 8 carbon atoms and no more than 34 carbon atoms. Dis a receptor ligand selected from the group consisting of receptorligand pairs biotin/avidin, peptide S/ribonuclease, complimentaryoligonucleotide pairs or antibody/ligand pairs, anddigoxigenin/anti-digoxigenin antibody.

In one embodiment of the present invention wherein thespectrophotometrically quantifiable linker is bound to a biomolecule viaa amino, thiol or carbohydrate reactive moiety and wherein thebiomolecule is selected from the group consisting of a protein, apeptide, an oligonucleotide and a polynucleotide. Alternatively thespectrophotometrically quantifiable linker may be bound to a biomoleculevia receptor ligand pairs such as biotin/avidin, peptide S/ribonuclease,digoxigenin/anti-digoxigenin antibody complimentary oligonucleotidepairs or antibody/ligand pairs. Correspondingly, a first biomolecule maybe bound via an amino, thiol or carbohydrate reactive moiety and asecond biomolecule may be bound via a receptor ligand pair to thespectrophotometrically quantifiable linker.

In another aspect of the present invention a method of preparing aspectrophotometrically quantifiable linker is provided by the steps ofpreparing a first conjugate of a first biomolecule bound to oneprofluorescent/prochromophoric compound of a fluorescent pair via anamino, thiol or carbohydrate reactive moiety and preparing a secondconjugate of a second biomolecule bound to a flexible linker via abiotin or a receptor ligand and the other profluorescent/prochromophoriccompound of a fluorescent pair and combining the first conjugate withthe second conjugate for a time thereby forming a hydrazone bond betweenthe profluorescent/prochromophoric compound pair forming a fluorescentmoiety.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: A diagrammatic representation of the chemistry for the formationof fluorescent hydrazones from conjugationally extended aldehydes andhydrazines;

FIG. 2: A diagrammatic representation of the tautomerization ofbis-(2-heteroaromatic)hydrazone chelates;

FIG. 3: Hydrazine and aldehyde succinimidyl ester reagents, SANH and SFBrespectively, developed for modification of amino moieties onbiomolecules and a diagrammatic representation of the conjugation of ahydrazine-modified biomolecules with a benzaldehyde-modifiedbiomolecule;

FIG. 4: (A) PAGE gel of the results of the conjugation of a5′-benzaldehyde-modified oligonucleotide to a hydrazine-modifiedantibody visualized by coomassie blue (CB) staining; (B) the same gelvisualized by UV backshadowing to visualize the oligonucleotideconjugated to the protein; (C) nitrocellulose membrane of the blottedconjugate following hybridization of the fluorescein-labeledcomplementary oligonucleotide demonstrating retention of hybridizationfunctionality of conjugated oligonucleotide;

FIG. 5: A diagrammatic representation of fluorescent hydrazone (3)formed from 6-hydrazinonicotinic acid (1; R═OH) and4-dimethylaminocinnamaldehyde (2);

FIG. 6: (A) Chemical structure of benzaldehyde phosphoramidite used toincorporate benzaldehyde moieties on the 5′-terminus of oligonucleotidesduring their solid phase synthesis; (B) PAGE gel of purifiedoligonucleotide (Lane 1) and the product of the reaction of theoligonucleotide with trans-4-hydrazinostilbazole (1; Fluka ChemicalCo.);

FIG. 7: Absorbance and emission spectra of a 22mer oligonucleotidemodified on the 5′-end with the hydrazone formed from the reaction ofbenzaldehyde and, trans-4′-Hydrazino-2-stilbazole;

FIG. 8: Chemical structure of bifunctional hydrazido amine modificationreagent SHTH;

FIG. 9: A diagrammatic representation showing hydrazones prepared fromconjugationally extended hydrazines and aldehydes form fluorescentspecies while hydrazones prepared from conjugationally extendedhydrazides and aldehydes do not form substantially fluorescent species.5′-(6-Hydrazinylpyridine)-modified oligonucleotide is reacted with4-dimethylaminocinnamaldehyde (Reaction A) andnaphthalene-1,2-dicarboxaldehyde (Reaction B) form fluorescent species.The hydrazone formed from the reaction of5′-(6-hydrazidoterephalate)-modified oligonucleotide with4-dimethylaminocinnamaldehyde is not fluorescent and the product withNDA forms a weakly fluorescent species based on the pyrollo-fusednaphthalene product without conjugation through hydrazide moiety;

FIG. 10: A diagrammatic representation of the conversion of cytidine to4-N-aminocytidine with hydrazine/bisulfite;

FIG. 11: A diagrammatic representation of the incorporation offluorescence into DNA wherein salmon sperm DNA was treated withhydrazine/bisulfite to convert cytidine moieties to 4-aminocytidine, anaromatic hydrazine. The modified DNA was treated withdimethylaminocinnamaldehyde (DAC; top reaction; Lane 2) ornaphthalene-1,2-dicarboxladehyde (NDA; bottom reaction; Lane 4) andvisualized following electrophoresis on an agarose gel (at left).Control reactions wherein untreated DNA was reacted with DAC and NDAwere not fluorescent (Lanes 1 and 3 respectively);

FIG. 12: Chemical structure of commercially available aromatichydrazines;

FIG. 13: Chemical structure of commercially available aldehydes;

FIG. 14: Chemical structure of cyanine dyes Cy3 and Cy5;

FIG. 15: Chemical structure of cyanine profluors and their parentfluorophores targeted for synthesis;

FIG. 16: A diagrammatic representation of the synthetic methods for thepreparation of hydrazinoheterocyles; and

FIG. 17: Chemical structure of benzimidazole profluors and synthesisschemes of their parent fluorophores targeted for synthesis.

FIG. 18: Chemical structure of biotin;

FIG. 19: A diagrammatic representation showing hydrazones prepared fromconjugationally extended hydrazines and aldehydes that form fluorescentspecies while hydrazones prepared from conjugationally extendedhydrazides and aldehydes do not form substantially fluorescent species.5′-(6-Hydrazinylpyridine)-modified oligonucleotide is reacted with4-dimethyl-aminocinnamaldehyde (Reaction A) andnaphthalene-1,2-dicarboxaldehyde (Reaction B) form fluorescent species.The hydrazone formed from the reaction of5′-(6-hydrazidoterephalate)-modified oligonucleotide with4-dimethylaminocinnamaldehyde is not fluorescent and the product withNDA forms a weakly fluorescent species based on the pyrollo-fusednaphthalene product without conjugation through hydrazide moiety;

FIG. 20: A schematic representation of the synthesis of amino-reactivebiotin/hydrazone chromophore 6;

FIG. 21: A graph showing amino-reactive biotin/hydrazone chromophore 6and overlaid spectra of equivalent amounts (20 μg) native bIgG and bIgGmodified with 5×, 10× and 15× amino-reactive biotin/hydrazonechromophore demonstrating the incorporation of chromophore/PEG4/biotinmoiety by their absorbency at A354.

FIG. 22: Structure of a thiol-reactive chromophore linker of the presentinvention (7), aldehyde-reactive chromophore linker of the presentinvention (8) and an oxidized carbohydrate-reactive chromophore linkerof the present invention (9);

FIG. 23: A schematic representation of the incorporation of aconjugationally extended aldehyde cytosine triphosphate 10 in a DNAamplicon (R═H) or RNA amplicon (R═OH) and labeling the modified ampliconwith a linker of the present invention 11 and

FIG. 24: Schematic representation of the synthesis of a linker of thepresent invention (11).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all terms used herein have the same meaning asare commonly understood by one of skill in the art to which thisinvention belongs. All patents, patent applications and publicationsreferred to throughout the disclosure herein are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail.

The term “biomolecule” as used herein refers to a compound of biologicalorigin, or of biological activity, that may have, or may be modified tohave, an amine group or carbonyl group that may be harnessed in theformation of a hydrazone bond with a novel carbonyl profluorophore ornovel hydrazine profluorophore of the present invention. Biomoleculesinclude for example a nucleic acid, a nucleotide, a protein, an aminoacid, a carbohydrate monomer and a polysaccharide. If the biomolecule isa nucleic acid it may be DNA, cDNA, RNA, or PNA and may comprise naturalor unnatural bases or internucleotide linkages such as for examplephosphodiesters, phosphorothioates, phosphoramidites or peptide nucleicacids.

The term “profluorophore” as used herein refers to a compound that may,or may not fluoresce, but when joined with its correspondingprofluorophore pair compound produces a fluorescent hydrazone compoundthat has a peak emission wavelength substantially separate from the peakemission wavelength of either of the profluorophores that they may thatmake up the fluorescent hydrazone compound. A profluorophore paircomprises a hydrazine-based profluorophore and a carbonyl-basedprofluorophore that when combined form a fluorescent hydrazone compound.

The term “pro-chromophore” as used herein refers to a compound that may,or may not produce a visible color, but when joined with itscorresponding pro-chromophoric pair compound produces a chromophoriccompound that has a peak observable wavelength substantially separatefrom the peak observable wavelength of either of the prochromophoresthat make up the chromophoric hydrazone compound. A pro-chromophoricpair comprises a hydrazine-based pro-chromophore and a carbonyl-basedpro-chromophore that when combined form a chromophoric hydrazonecompound.

The term “reactive linking moiety” as used herein refers to moleculesused commercially for binding one molecule to another based on thepresence of a particular chemical group on the molecule of interest.Some commercially sold molecules referred to herein as linking moietiesinclude those that react with free amines on the target molecule, suchas N-hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl andN-hydroxybenzotriazolyl ester and those that react with free sulfhydrylspresent on the target molecule such as maleimido, α-haloacetamido andpyridyldisulfides.

The term “ligand/receptor couple” as used herein refers to a pair ofmolecules having a substantially high affinity of binding specificallyto one another. One example of such a binding pair would be a receptoron a cell and the ligand that binds that receptor. Another example wouldbe biotin and avidin, which are two molecules that have a strongaffinity for binding each other having an association constant of around10¹⁵. Other pairs include Peptide S and ribonuclease A, digoxigenin andit receptor and complementary oligonucleotide pairs.

To achieve the optimal signal from a fluorescent label it is importantthat the structural integrity of the fluorophore is retained throughoutprocessing of the labeled reporter molecule. A disadvantage withcommercially available fluorophores is their propensity to behydrolytically unstable or photobleach. The ability to efficiently formfluorescent species in situ in biological media in contrast to presentmethods wherein a labile fluorescent species is present throughout allprotocols would be extremely advantageous in yielding products withfully retained fluorescence for improved limits of detection. In onecurrent example in DNA microarrays, fluorescently labeled triphosphates,e.g. Cy3 and Cy5 triphosphosphates (Amersham Biosciences, Piscataway,N.J.), are incorporated during PCR or reverse transcriptaseamplification however quenching of the fluorophores throughphotobleaching or hydrolysis occurs during the many manipulationsrequired to isolate the desired fluorescently labeled polynucleotide. Toovercome this problem a less than ideal two-step method has beendeveloped wherein a 3-aminoallylcytidine triphosphate is incorporatedduring polynucleotide amplification with subsequent purification,labeling with fluorescent succinimidyl esters and final purification toremove excess unincorporated fluorescent molecules. This chemistry isbased on a amino/succinimidyl ester reaction that requires large excessof succinimidyl ester due to its instability in water and steps toremove the excess hydrolyzed reagent. This reaction proceeds over asmall pH range, i.e. 7.2-8.0 and is concentration dependent.

It would be advantageous to have a method wherein a stablenon-fluorescent species is used to label a biomolecule that followingall required processing in techniques such as PCR, 2-dimensionalelectrophoresis or immunohistochemistry can be reacted efficiently witha second non-fluorescent molecule to form a fluorescent species. Thepresent invention describes a chemistry wherein a conjugationallyextended hydrazine reacts with a conjugationally extended carbonyl insitu in aqueous media to form a fluorescent molecule (FIG. 1). Bothaldehydes and hydrazines are stable in aqueous media and reactefficiently to form stable hydrazones. The hydrazone formation is acidcatalyzed and has an optimum pH of 4.7 but proceeds up to pH 8.0. Thismethodology could be extended to use with biosensors for biowarfare andpathogen detection, brand security and Near-IR products. Thesefluorophores may also be engineered for use in laser and photonicsapplications.

D. E. Ryan, F. Snape and M. Winpe (Ligand Structure and Fluorescence ofMetal chelates; N-Heterocyclic Hydrazones with Zinc, Anal, Chim. Acta58:101, 1972) described a series of hydrazone chelates (Table 1) andthat upon addition of Zn²⁺ the chelates complex the metal yielding afluorescent metal chelate (FIG. 2). It was postulated how thenon-complexed chelate can exist in two different tautomers that havedifferent fluorescent properties due to disrupted aromatic bonding. Theaddition of the zinc ion ‘locks in’ the tautomer with better conjugationand higher fluorescence. These authors further described the use ofthese chelates as analytical tools for determination of trace amounts,i.e. parts per million and parts per billion, of zinc.

Abbreviated Relative Full Name Form λ_(excitation) λ_(emission)Fluorescence* Pyridine-2-aldehyde-2-pyridyl hydrazone PAPH 455 515 1Quinoline-2-aldehyde-2-pyridylhydrazone QAPH 490 540 2Phenanthridine-2-aldehyde-2-pyridylhydrazone PDAPH 490 545 7Pyridine-2-aldehyde-2-quinolylhydrazone PAQH 470 535 660Quinoline-2-aldehyde-2-quinolylhydrazone QAQH 495 595 30Phenanthridine-2-aldehyde-2- PDAQH 525 610 16 quinolylhydrazonePyridine-2-aldehyde-2- PAPDH 450 540 100 phenanthrdinylhydrazoneQuinoline-2-aldehyde-2- QAPDH 510 600 110 phenanthrdinylhydrazonePhenanthridine-2-aldehyde-2- PDAPDH 580 620 230 phenanthrdinylhydrazoneBenzimidazole-2-aldehyde-2-pyridylhydrazone BAPH 440 510 140470B550enzimldazole-2-aldehyde-2- BAQH 470 520 2000 quinolylhydrazoneBenzimidazole-2-aldehyde-2- BAPDH 480 530 440 phenanthrdinylhydrazonePhenyl-2-pyridylketone-2-pyridylhydrazone PPKPH 420 470 8Phenyl-2-pyridylketone-2-quinolylhydrazone PPKQH 470 550 450Phenyl-2-pyridylketone-2- PPKPDH 490 575 1520 phenanthrdinylhydrazoneTable 1 lists the bis-(2-heteroaromatic)hydrazones prepared by Ryan etal, supra. and including their excitation and emission wavelengths andrelative fluorescence properties.

Bifunctional hydrazine and carbonyl reagents to modify biomolecules havebeen prepared. FIG. 3 outlines this chemistry. The hydrazine/carbonylbioconjugation couple has significant advantages over currently usedmaleimido/thiol couple in that both the aldehyde and hydrazine moietiesare stable following incorporation on biomolecules, simple addition ofan aldehyde-modified biomolecule to a hydrazine-modified biomoleculeyields a stable hydrazone without the requirement of a reductionreaction to stabilize the bond, the stability of the functional groupsallows conjugations to be performed at low concentrations, i.e. <100microgram/mL and the chemistry has been engineered to prepare conjugatesfrom all biomolecules.

FIG. 4 shows the conjugation of an 5′-[4 formalbenzamide]-modifiedoligonucleotide to a hydrazine-modified antibody. The resultsdemonstrate complete conversion of modified protein to conjugate by thesimple addition of the stable 5′-[4 formalbenzamide]-modifiedoligonucleotide to the modified-hydrazine modified protein forming astable hydrazone mediated conjugate.

The linkers have been prepared as reagents for the solid phase synthesesof peptides (hydrazino carboxylic acids) and oligonucleotides (aldehydephosphoramidites). Aldehyde-modified deoxy and ribo-triphosphates havealso been prepared and demonstrated to be incorporated intopolynucleotide amplicons.

In the initial demonstration of the fluorescence of conjugationallyextended hydrazones, 6-hydrazinonicotinic acid (1) (SolulinkBiosciences, San Diego, Calif.) was reacted with4-dimethylcinnamaldehyde (2) (Aldrich Chemical Co., Milwaukee, Wis.) toyield fluorescent hydrazone (3) (FIG. 5). Hydrazone (3) absorbed at 397nm and emitted at 508 nm a Stokes shift of 109 nm. Other hydrazonesprepared from commercially available conjugationally extended hydrazinesand aldehydes were prepared and their respective excitation and emissionwavelengths are presented in Table 2 below. It should be noted that theStokes shifts for hydrazones 2, 3 and 4 all are 100 nm or greater.

absorbance emission nm nm

385 407

355 472

397 508

450 550Table 2 shows the fluorescent hydrazones and their absorbance andemission maxima.

In another demonstration benzaldehyde phosphoramidite has been preparedthat is used to incorporate benzaldehyde moieties directly on the 5′-endof oligonucleotides during solid phase oligonucleotide synthesis. Theincorporation of this moiety is accomplished with similar identicalprocedures and yields as incorporation of DMT-amino modifiedphosphoramidites. Reaction of an oligonucleotide withtrans-4′-hydrazino-2-stilbazole dihydrochloride quantitatively yields afluorescent oligonucleotide (FIG. 6). The emission and absorbancespectra of hydrazone (4) (see Table 2 above) linked to a 22meroligonucleotide are presented in FIG. 7.

Methods have been developed to prepare both hydrazino- andhydrazido-modified oligonucleotides. Hydrazinopyridine-modifiedoligonucleotides can be prepared by the reaction of amino-modifiedoligonucleotides with SANH and hydrazido-modified oligonucleotides canbe prepared using SHTH (FIG. 8). To demonstrate that hydrazones preparedfrom conjugationally extended hydrazines but not conjugationallyextended hydrazides both oligonucleotides were reacted with4-dimethylaminocinnamaldehyde (FIG. 9, reactions A and C) but only thehydrazine derived hydrazone was fluorescent. In another demonstrationboth hydrazino- and hydrazido-modified oligonucleotides were reactedwith 1,2-naphthalene-dicarboxaldehyde (NDA; reactions B and D). It isknown that amines react with NDA yield a fluorescent species. Theproducts from the reaction of these oligonucleotides were bothfluorescent however the hydrazine derived product absorbed and emittedqualitatively more intensely and at longer wavelengths than thehydrazido-modified oligonucleotide.

In another demonstration salmon sperm DNA was treated withhydrazine/bisulfite to convert cytidine moieties to 4-N-aminocytidine,an aromatic hydrazine (FIG. 10; Negishi, K., Harada, C., Ohara, Y.,Oohara, K., Nitta, N. and Hayatsu, H., 4-N-aminocytidine, a nucleosideanalog that has an exceptionally high mutagenic activity, Nucleic AcidsRes. 1983, 11, 5223-33)). The reaction of the modified DNA with both4-dimethylaminocinnamaldehyde and naphthalene-1,2-dicarboxaldehyde (NDA)yielded fluorescent DNA. (FIG. 11).

It should be noted that the hydrazine-modified cytidine is a componentof the fluorophore and not solely a linkage point. It is anticipatedthat conjugationally extended aldehydes that yield hydrazones with moreintensely fluorescent properties can be developed to convert reversetranscribed DNA to fluorescent species thereby using all naturaltriphosphates in the reverse transcription reaction and not substitutedtriphosphates whose incorporation is random and not quantitativelyreproducible batch to batch.

A library of hydrazone fluorophores may be prepared from commerciallyavailable aromatic hydrazines and aldehydes using the methods described.FIG. 12 below presents structures of commercially available hydrazinesthat will be purchased to be reacted to form hydrazone fluorophores.

FIG. 13 presents structures of commercially available aldehydes thatwill be purchased to be reacted to form hydrazone fluorophores.

The initial pro-fluorophore structures targeted for syntheses in thisprogram are based on cyanine dyes. These dyes are extremely sensitiveand have been developed for a variety of commercial uses including lifesciences applications as well as photographic uses (A. Mishra, R. K.Behera, P. K. Behera, .K. Mishra and G. B. Behera, Cyanines during the1990's: A Review, Chem. Rev., 100:1973, 2000). FIG. 14 below presentsthe structures of the most used cyanine dyes, Cy3 and Cy5, for lifescience applications. These dyes are routinely used as reportermolecules in both gene and protein microarrays.

FIG. 15 presents aldehyde and hydrazine cyanine-based profluorophoresand their parent fluorophores targeted for synthesis.

Two methods have been developed for the preparation ofhydrazino-substituted aromatic compounds (FIG. 16). The classical methodfor the synthesis of 2-hydrazinoheteroaromatic compounds is directnucleophilic aromatic substitution of 2-chloro-heterocycles withhydrazine. Arterburn et al. (J. B. Arterburn, K. V. Rao, R. Ramdaa andB. R. Dible, Org. Lett, 2001, 3, 1351 and J. B. Arterburn, B. D. Bryantand D. Chen, Chem. Comm. 2003, 1890) have developed palladium-catalyzedprotocols to convert 2-substituted bromo, chloro and trifluorosubstituted pyridines to 2-hydrazinylpyridines.

Aromatic aldehydes can be prepared by a variety of methods includingdirect oxidation of methyl-substituted aromatic moieties and reductionof aromatic nitriles. Aromatic aldehydes can be conjugationally extendedusing the Mannich reaction.

Due to the fluorescence of benzimidazole-quinoline hydrazone (5) avariety of pro-fluorophores based on this parent core structure havebeen investigated. FIG. 17 presents target pro-fluorophores and theirrespective parent fluorophores.

Diverse libraries with varying fluorescent properties can be readilyprepared as any carbonyl and any hydrazine prepared or commerciallyavailable can be combined to yield a fluorescent hydrazone. Theexcitation and emission characteristics desired can be tailored byincorporation of substituents such as dimethylamino, alkoxy and nitrogroups.

The photophysical characteristics of the fluorophores may be observedusing a QM-2 Spectrofluorimeter (Photon Technologies International,Inc.), with a nitrogen-dye laser/second harmonic generator excitationsource. A Xe arc lamp may be utilized having excitation that allows forthe collection of steady state excitation and emission spectra, thecharacterization of quantum yield, photo-bleaching, and an degradationof fluorescence from these species. The response of this instrument maybe characterized by fluorescence quantum yield standards (i.e. quininesulfate) to determine the quantum yield of the various fluorophores. Thelaser system with the laser-strobe detection attachment allows for thecollection of sub nanosecond time-decays. The time decay curves may beanalyzed to determine the excited-state lifetimes of these fluorophores.

In addition a Nd:YAG laser pumped OPO system, will allow for tunableexcitation between 400 nm and 3000 nm. The detection system includes aJobin-Yvon 0.5 m monochromator with both PMT and CCD detection. The CCDcamera is sensitive in the visible and Near Infrared regions of theelectromagnetic spectrum. This system may be used for thecharacterization of fluorophores in the far-red region of the visiblespectrum and in the NIR region. The tunable excitation will provide ameans to excite fluorophores, regardless of their absorption spectra inthe visible/NIR regions

The stability of the commercially available fluorophores has limited thefull range of development of a variety of applications. The advantageouscharacteristics of this technology includes: elimination of the need toremove the excess second moiety from the in situ formed fluorescentspecies as it is either not fluorescent or has completely differentfluorescent properties that do not interfere with detection of the newfluorescent species; increased efficiency of the formation of thefluorescent species >90%, in buffered aqueous media, pH 5.0-8.0.; theability to prepare a wide variety of fluorophores of differentabsorbance and emission wavelengths by varying the structures of the twomoieties of the final fluorescent molecule; utilizing a linker moietythat may be incorporated on either of the pro-fluorescent species forcovalent linking to a biomolecule or a surface; significant reduction inphotobleaching or increased hydrolytic stability of the initialpro-fluorophore as has it will be in a lower energy state than fullyconjugated fluorophores currently employed; and the development offluorescent species having well separate spectral absorbance andemission properties, i.e. a Stoke's shift >100 nm.

U.S. patent application Ser. No. 60/546,104 to Schwartz incorporatedherein in its entirety has described the in situ preparation ofhydrazone fluorophores by the reaction of a conjugationally extendedaldehyde with a conjugationally extended hydrazine one of which islinked to biomolecular probe such as an antibody or an oligonucleotide.FIG. 19 presents the reaction scheme for the reaction of aconjugationally extended hydrazine with a conjugationally extendedaldehyde linked to an oligonucleotide forming an oligonucleotide linkedfluorescent hydrazone. The scheme also presents results thatdemonstrated that the reaction is specific for a conjugationallyextended hydrazine and not a hydrazide. In contrast to formingchromophore/fluorophores in situ the present invention incorporates apre-formed chromophoric/fluorescent hydrazone into the linker comprisingthe ligand for direct spectrophotometric quantitation of the level ofincorporation of the ligand when bound to a biomolecule such as aprotein or nucleic acid.

FIG. 20 presents the construction of an amino-reactive biotin moietythat has incorporated in its chain a chromophoric hydrazone forspectrophotometric quantitiation and a short PEG linker that is requiredto retain the binding affinity of biotin to streptavidin. Thistri-functional molecule can be readily quantified spectrophotometricallyfollowing conjugation to a biomolecule because of its unique molarextinction coefficient (generally >20000) and its unique absorbance orfluorescence (generally at wavelengths greater than 300 nm and atfrequencies having no, or only minimal, observable signals prior toconjugation). It is anticipated that more highly conjugated systems thanpresented in FIG. 20 will absorb at longer wavelengths with greaterextinction coefficients or fluorescence allowing even greatersensitivity. FIG. 21 presents constructions of thiol and oxidizedcarbohydrate-reactive linkers of the present invention.

The incorporation of labels into nucleic acids such as cDNA or cRNAusing polymerases and reverse transcriptases respectively for geneexpression analysis by microarrays is a multi-step procedure thatrequires high levels of reproducibility so results can be reliablycompared between experiments. One current method for labeling cDNA orcRNA is the use of a nucleoside modified to incorporate a biotinmolecule on the minor groove side. One of the most commonly used methodsto label and detect labeled cDNA and cRNA is using a biotinylatednucleoside triphosphate (NTP). As there are only labor-intensive methodsto quantitate the level of biotin incorporation in the amplicon, thebiotin-modified amplicon is used directly without quantitation. It wouldbe extremely advantageous to be able to directly quantitate the level ofbiotin incorporated into cDNA or cRNA. FIG. 22 is a schematic diagram ofthe synthesis of a nucleoside triphosphate modified with aconjugationally extended aldehyde such as a benzaldehyde moiety and tolabel the amplicon after elongation by reaction with a biotinylatedconjugationally extended hydrazine. U.S. Pat. No. 6,686,461 to D.Schwartz and R. Hogrefe which is incorporated herein by reference in itsentirety more fully discloses this synthesis. The chemistry describedherein is advantageous in that the formation of the hydrazone is highyielding at near stoichiometric amounts, a chromophore is formed thatwill allow batch-to-batch quantitiation of levels of incorporation ofbiotin and a short polyethylene linker is incorporated is necessary toretain the affinity of the biotin to its cognate receptor avidin.

In another protocol the amplicon may be hybridized prior to reactionwith the biotin hydrazide and subsequently detected with afluorescently-labeled avidin or anti-biotin antibody. Thebenzaldehyde-labeled amplicon can be quantitated by removing an aliquotand treating it with a hydrazide pro-fluorophore to form a fluorescenthydrazone and spectrophotometrically quantitating the level of aldehydeincorporation. This may be advantageous as the hybridization reactionwill have minimal modification resulting in less sterically encumberedhybridization.

In use the linker moiety reacts with a biomolecule such as an antibodyunder appropriate reaction conditions. The conjugate is then purifiedand the protein concentration determined. The number of biotinmolecules/protein molecule is determined by observing the absorbance ofa known concentration of the conjugate in solution at a wavelength>300nm. The concentration of the chromophore and therefore the biotin isdetermined by dividing the absorbance reading by the extinctioncoefficient of the chromophore incorporated in the chain. Thisconcentration is divided by the mM concentration of the protein and thenumber of biotin molecules per conjugated is determined.

EXAMPLES Example 1 Synthesis of Biotin/PEG/hydrazone succinimidyl ester6 (FIG. 20)

PMR spectra were obtained on a Bruker 500 MHz NMR at NuMega Laboratories(San Diego, Calif.) and electrospray mass spectral data was obtained atHT Laboratories (San Diego, Calif.),

1. Synthesis of Mono-Boc-1,13-diamino-4,7,10-trioxatetradecane (1;(3-{2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy}-propyl)-carbamic acidtert-butyl ester), Amine 1

To a solution of 4,7,10-trioxa-1,13-tridecanediamine (FIG. 20) (30 g;mmol) in dichloromethane (1000 mL) was added a solution of di-t-butyldicarbonate (10 g; mmol; Aldrich Chemical Co., Milwaukee, Wis.) indichloromethane (200 mL) over 2 h. The reaction mixture was stirred atroom temperature for 4 hours. Thin layer chromatography (TLC, silicagel) using dichloromethane/methanol/triethylamine (90/10/1); ninhydrindevelopment) indicated the presence of two new spots, a minor spot at Rf0.8 ascribed to the bis-BOC product and a major spot at Rf (0.2) for thedesired product. The reaction mixture was washed with water (4×500 mL)to remove the excess diamine and the organic phase was dried overmagnesium sulfate, filtered and concentrated to give a viscous oil thatwas purified by flash chromatography over silica gel using DCM/MeOH/TEA(95/5/1) to give 10.5 g of desire Amine 1 as an oil.

2. Synthesis of((3-{2-[2-(3-{[6-(N′-Isopropylidene-hydrazino)-pyridine-3-carbonyl]-amino}-propoxy)-ethoxy]-ethoxy}propyl)-carbamicacid tert-butyl ester), Hydrazone 2

To a solution of Amine 1 (1.05 g; 3.28 mmol) in DCM (20 mL) was added asolution of succinimidyl 6-hydrazinonicotiniate acetone hydrazone (0.951g; 3.28 mmol; Solulink Biosciences, Inc., San Diego, Calif.) in DCM (10mL). The reaction mixture was stirred at room temperature for 6 hours.Subsequently the reaction mixture was washed with water and brine. Theorganic phase was dried (magnesium sulfate), filtered and concentratedto give 1.2 g of Hydrazone 2 as a colorless thick oil.

3. Synthesis of(4-{[5-(3-{2-[2-(3-tert-Butoxy-carbonylamino-propoxy)-ethoxy]-ethoxy}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzoicacid), Hydrazone 3

To Hydrazone 2 (0.405 g: 0.81 mmol) in MeOH (5 mL) and 100 mM MES, 150mM NaCl (5 mL) was added a solution of 4-carboxybenzaldehyde (0.121;0.81 mmol) in MeOH (3 mL). The reaction mixture is allowed to stir atroom temperature overnight. Copious precipitate formed. The reactionmixture was centrifuged and the solids were washed with a 1/1 solutionof MeOH/MES. The solids were dried under vacuum to yield 0.42 g ofHydrazone 3 as a pale yellow solid and used directly in the next step.

4. Synthesis of(4-{[5-(3-{2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzoicacid hydrochloride salt), chromophore Hydrazone 4

A solution of Hydrazone 3 (0.388 g; 0.66 mmol) in dioxane (15 mL) wasprepared with heating. The solution was cooled to room temperature and 4N HCl in dioxane (4 mL; Aldrich Chemical Co., Milwaukee, Wis.) was addedsuccinimidyl and the reaction was stirred at room temperature for 16 h.A precipitate formed on stirring. The reaction mixture was centrifugedand the solids were washed with dioxane (3×10 mL). The solids wereresuspended in dioxane and concentrated under vacuum to yield 240 mg ofamino/PEG4/Hydrazone 4 as a pale yellow solid. Electrospray mass spec:expected m/e 487. found positive mode 488 (M+H), negative mode 486 (M−H)and 522 (M+Cl⁻).

5. Synthesis of Biotin/PEG4/chromophore succinimidyl ester 6(5-(N′-{4-[2-(2,5-Dioxo-pyrrolidin-1-yl)-2-oxo-acetyl]-benzylidene}-hydrazino)-pyridine-2-carboxylicacid{3-[2-(2-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-propoxy}-ethoxy)-ethoxy]-propyl}-amide)

To a solution of amino/PEG4/hydrazone 4 (0.780 g; 1.60 mmol) in DMF (25mL) was added biotin succinimidyl ester (0.546 g; 1.60 mmol) followed bythe addition of triethylamine (0.726 mL; 4.80 mmol). The solution wasstirred at room temperature until complete as determined by silica gelTLC using DCM/MeOH/TEA (90/10/1) as eluant (developed by UV to visualizethe pyridine chromophore and dimethylaminocinnamaldehyde/sulfuricacid/ethanol spray followed by heating to visualize the biotin moiety).To the reaction mixture N-hydroxysuccinimide (0.184 g; 1.60 mmol) andDCC (0.330 g; 1.60 mmol) were added and stirred at room temperature for16 hours. The reaction mixture was concentrated to dryness andpartitioned between DCM and water. The organic phase was further washedwith brine, dried (magnesium sulfate), filtered and concentrated to givea yellow sticky solid. The solids were triturated with ethyl acetate.The solids were isolated by filtration to give 830 mg of a yellow solid.TLC (DCM/MeOH/TEA (90/10/1) indicated one major spot (visualized by UVand dimethylaminocinnamaldehyde/sulfuric acid/ethanol solution) and HPLCanalysis (YMC C-18, 150×4.6 cm; 5 □m; 120 A; gradient mobile phase A:water/acetonitrile/trifluoroacetic acid (20/80/0.1), mobile phase B:0.1% TFA in water; gradient 10% A/90% B to 100% A over 20 min; retentiontime 8.8 min, detection @A254 and A350. PMR (DMSO-d₆) δ: 11.64, s (1H),8.65, d, (1H), 8.37 t, (1H) NH, 8.12 dd (1H), 7.95 and 8.11 ab system(4H), 7.73 t (1H) NH, 7.36 d (1H), 6.41 s (1H), 6.35 s (1H), 5.57 d(1H), 4.29 br. t (1H), 4.11 br. t (1H), 3.3-3.55 m (12H), 3.08 m (4H),2.90 s (4H), 2.88 dd (1H), 2.57 d (1H), 2.03 t (2H), 1.75 m (2H), 1.59 m(2H), 1.2-1.5 m (8H). The extinction coefficient ofBiotin/PEG4/chromophore succinimidyl ester 6 was determined bydissolving Biotin/PEG4/chromophore succinimidyl ester 6 (1.0 mg) in DMF(1 mL) and diluting into PBS. The absorbance maximum was A354 and themolar extinction coefficient was determined to be 23,250.

Example 2 Protein labeling with Biotin/PEG4/chromophore/succinimidylester 6

Bovine immunoglobulin (bIgG; Sigma Chemical Co., St. Louis, Mo.) wasdissolved in modification buffer (100 mM phosphate, 150 mM NaCl, pH 7.2)to prepare a 5 mg/mL solution. A solution ofBiotin/PEG4/chromophore/succinimidyl ester 6 (1 mg) dissolved in DMF(100 mL) was prepared. Three separate reactions were performed wherein 5mole equiv., 10 mol equiv. and 15 mol equiv. ofBiotin/PEG4/chromophore/succinimidyl ester 6 (1.3, 2.6 and 3.9 μL,)respectively were added to 0.5 mg bIgG solution. The reaction wasallowed to incubate at room temperature for 2 hours. The reactionmixtures were desalted into PBS using Biomax diafiltration apparatuses(Millipore, Inc., Bedford, Mass.). Protein concentrations of all themodified proteins were determined using the BCA assay (Pierce ChemicalCo., Rockford, Ill.). Spectral analyses of each product were performedby diluting 20 mg of modified protein to 100 mL in PBS. The number ofmoles of chromophore incorporated was calculated by determining theabsorbance of the protein at A354 dividing by the molar extinctioncoefficient, i.e. 29000, of the chromophore. The overlaid spectra of theproducts as well as unmodified IgG are present in FIG. 21A. The numberof incorporated biotins in the modified proteins was further analyzed bythe HABA assay (Pierce Chemical Co., Rockford, Ill.). The results, bothtabular and graphically, from both the UV spectral assay and the HABAassay are presented below.

IgG/HABA IgG/A354  5X 1.03 2.45 10X 1.60 4.71 15X 2.22 6.25A further experiment to demonstrate retention of binding activity of thechromophore/biotinylated bIgG the modified proteins were incubated withstreptavidin and the reaction products were analyzed by PAGE gelelectrophoresis. FIG. 21B presents the results.

Example 3 Synthesis of Biotin/PEG/hydrazone 10 (FIG. 24) 1. Synthesis of({3-[2-(2-{3-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-propoxy}-ethoxy)-ethoxy]-propyl}-carbamicacid tert-butyl ester),1-biotinamido/PEG/BOC-amino 14

To a solution of Amine 1 (0.544 g; 1.70 mmol) in DMF (15 mL) was added asolution of biotin succinimidyl ester (0.580 g; 1.70 mmol) in DMFfollowed by the addition of TEA (0.75 mL; 5.09 mmol). The reactionmixture was stirred at room temperature for 16 h. The solvent wasremoved on the rotavap and the residue was partitioned between DCM andwater. The organic phase was further washed with brine, dried (magnesiumsulfate), filtered and concentrated to give 415 mg of1-biotinamido/PEG/BOC-amino 14 as an amorphous solid. The product was asingle spot by TLC (DCM/MeOH/TEA (90/10/1); developed bydimethylcinnamaldehyde/ethanol/sulfuric acid/heat to visualize thebiotin moiety). The product was used directly in the next step.

2. Synthesis of(5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid(3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propyl)-amide),1-biotinamido/PEG/amino 15

To a solution of 1-biotinamido/PEG/BOC-amino 14 (400 mg; 0.73 mmol) wasdissolved in dioxane (20 mL) with mild heating. The solution was cooledto room temperature and a solution of 4 N HCl in dioxane (10 mL; AldrichChemical Co., Milwaukee, Wis.) was added. The reaction was stirred for14 h. The solvent was removed on the rotavap and the residue wasco-evaporated twice from dry dioxane. The product,1-biotinamido/PEG/amino 15, was used directly without purification.

3. Synthesis of (5-(N′-Methylene-hydrazino)-pyridine-2-carboxylic acid{3-[2-(2-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-propoxy}-ethoxy)-ethoxy]-propyl}-amide),1-biotinamido/PEG/amido-6-hydrazino-4-nicotinamide 11

To a solution of 1-biotinamido/PEG/amino 15 (0.375 g; 0.78 mmol) in DMF(25 mL) was added a solution of SANH (0.225 g; 0.78 mmol) andtriethylamine ((0.645 mL; 4.66 mmol)). The reaction mixture was stirredat room temperature for 16 h. The solvent was removed on the rotavap andthe residue was partitioned between DCM and water. The organic phase wasfurther washed with brine, dried (magnesium sulfate), filtered andconcentrated to give 290 mg of1-biotinamido/PEG/amido-6-hydrazino-4-nicotinamide 11 as an amorphoussolid. The product was a single spot, Rf 0.33, by TLC (DCM/MeOH/TEA(90/10/1) developed by dimethylcinnamaldehyde/ethanol/sulfuric acid/heatto visualize the biotin moiety). Mass spectral data: exptd m/e 621; posmod exptd m/e 622 (M+H). found 622 and exptd 644 (M+Na). found 644; negmode exptd m/e (M−H) 620. found 620 and (M+Cl⁻) 656. found 656

1. A fluorescent hydrazone compound of formula I,(R₁R₂)NN═C(R₁R₂)  I wherein: R₁ are independently a substituted orunsubstituted conjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ are independentlya hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, abranched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphaticmoiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine and wherein saidcomposition has an emission frequency equal to or greater than 400 nm.2. A fluorescent hydrazone compound according to claim 1 wherein one ofR, one of R₁ or one of R₂ further comprises a linker moiety selectedfrom the group consisting of an amino reactive moiety, a thiol reactivemoiety, an ester moiety and a modified carbohydrate monomer moiety.
 3. Afluorescent hydrazone compound according to claim 2 wherein said linkerfurther comprises a biomolecule.
 4. A fluorescent composition of theformula III,

wherein; R₁ (which is R₂) is a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ (which is R₃) is ahydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, abranched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphaticmoiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₃ (which is R₄) is Hor OH; R₄ (which is R₆) is H or a nucleic acid moiety; and R₅ (which isR₇) is PO₃ or a nucleic acid moiety.
 5. A profluorescent hydrazinecompound of formula IV,(R₁R₂)N(H)_(n)(NH₂)_(m)  IV wherein: R1 is a substituted orunsubstituted conjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ is a hydrogen, astraight chain aliphatic moiety of 1-10 carbon atoms, a branchedaliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10carbon atoms, a substituted or unsubstituted conjugationally extendedmoiety wherein the unsubstituted conjugationally extended moiety is analkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic orpolyheteroaromatic moiety and wherein the substituted conjugationallyextended moiety may be substituted with any combination of one or moreof the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo,unsubstituted amine and substituted primary, secondary, tertiary andquaternary amine; n is 0 when m is 2 and n is 1 when m is
 1. 6. Aprofluorescent hydrazine compound according to claim 5 wherein R₁ or R₂further comprise a linkable moiety selected from the group consisting ofan amino reactive moiety, a thiol reactive moiety, an ester moiety and amodified carbohydrate monomer moiety.
 7. A profluorescent hydrazinecompound according to claim 6 wherein said linker further comprises abiomolecule.
 8. A composition comprising a polymer having one or moreprofluorescent hydrazine compounds according to claim 5 bound to saidpolymer by one or more linker moieties.
 9. A polymer according to claim8 wherein said polymer is poly-lysine, poly-ornithine orpolyethyleneglycol.
 10. A profluorescent oxyamine compound of formulaVI,(R₁R₂)ONH₂  VI wherein: R₁ is a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; and R₂ is a hydrogen,a straight chain aliphatic moiety of 1-10 carbon atoms, a branchedaliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10carbon atoms, a substituted or unsubstituted conjugationally extendedmoiety wherein the unsubstituted conjugationally extended moiety is analkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic orpolyheteroaromatic moiety and tertiary and wherein the substitutedconjugationally extended moiety may be substituted with any combinationof one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro,carboxy, sulfo, unsubstituted amine and substituted primary, secondary,quaternary amine.
 11. A profluorescent oxyamine compound according toclaim 10 wherein R₁ or R₂ further comprise a linkable moiety selectedfrom the group consisting of an amino reactive moiety, a thiol reactivemoiety, an ester moiety and a modified carbohydrate monomer moiety. 12.A profluorescent oxyamine compound according to claim 10 wherein saidlinker further comprises a biomolecule.
 13. A method of preparing afluorescent hydrazone according to claim 1 by combining a hydrazine offormula IV,(R₁R₂)N(H)_(n)(NH₂)_(m)  IV with a carbonyl of formula V:O═C(R₁R₂)  V wherein: R₁ are independently a substituted orunsubstituted conjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; R₂ are independentlya hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, abranched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphaticmoiety of 1-10 carbon atoms, a substituted or unsubstitutedconjugationally extended moiety wherein the unsubstitutedconjugationally extended moiety is an alkenyl, alkynyl, aromatic,polyaromatic, heteroaromatic or polyheteroaromatic moiety and whereinthe substituted conjugationally extended moiety may be substituted withany combination of one or more of the groups hydroxy, alkoxy, alkene,alkyne, nitro, carboxy, sulfo, unsubstituted amine and substitutedprimary, secondary, tertiary and quaternary amine; n is 0 when m is 2and n is 1 when m is 1 for a time and under conditions that allowhydrazone formation.
 14. A method according to claim 13 wherein R₁ or R₂of formula IV further comprises a linkable moiety selected from thegroup consisting of an amino reactive moiety, a thiol reactive moiety,an ester moiety and a modified carbohydrate monomer moiety.
 15. A methodaccording to claim 13 wherein R₁ or R₂ of formula V further comprises alinkable moiety selected from the group consisting of an amino reactivemoiety, a thiol reactive moiety, an ester moiety and a modifiedcarbohydrate monomer moiety. 16.-24. (canceled)